Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo–keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/Km) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 Å (1 Å=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8–9 kJ/mol.
Skip Nav Destination
Article navigation
January 2006
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
December 12 2005
Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis
Regina Kratzer;
Regina Kratzer
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
Search for other works by this author on:
Stefan Leitgeb;
Stefan Leitgeb
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
†Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
Search for other works by this author on:
David K. Wilson;
David K. Wilson
†Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
Search for other works by this author on:
Bernd Nidetzky
Bernd Nidetzky
1
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
1To whom correspondence should be addressed (email bernd.nidetzky@tugraz.at).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
May 20 2005
Revision Received:
July 19 2005
Accepted:
August 22 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (1): 51–58.
Article history
Received:
May 20 2005
Revision Received:
July 19 2005
Accepted:
August 22 2005
Citation
Regina Kratzer, Stefan Leitgeb, David K. Wilson, Bernd Nidetzky; Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. Biochem J 1 January 2006; 393 (1): 51–58. doi: https://doi.org/10.1042/BJ20050831
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.